Dolichyl phosphates have been described as carriers of carbohydrate units in the biosynthesis of glycoproteins in animal tissues. The investigations proposed herein are designed to isolate and characterize dolichyl pyrophosphate synthetase and dolichol kinase. The mechanism of the formation of these unique polyprenol products will be of particular interest. In vivo and in vitro studies in several tissues, such as bovine liver, MOPC-315 cells, and chinese hamster ovary cells will be made to determine if mevalonic acid will serve as a precursor of dolichol. The kinases and synthetases will be solubilized from one or more of these tissues using methods such as detergent and salt extraction. These enzymes will be purified by a combination of fractionation techniques. The synthetase specificity, will be investigated with respect to the stereochemistry and chain length of the allylic pyrophosphate substrates. Purified dolichol kinase will be studied with respect to both alcohol and nucleotide substrate specificity. Comparison of other reaction requirements with those described for the analogous bacterial, undecaprenol, kinase will be of particular interest. Other important factors, such as detergent, phospholipid, and divalent cation requirements, will also be studied for both enzymes. The enzymically-produced polyprenyl pyrophosphates will be characterized after hydrolysis to the free polyprenols. These alcohols will be isolated and purified by reverse thin-layer chromatography and high-pressure liquid chromatography. The polyprenols will be characterized, with respect to both their chain length and stereochemistry, using a combination of techniques, such as mass spectroscopy, double label isotope experiments, and incorporation of radioactive label from stereospecifically tritiated delta3-23H-2R- and delta3-23H-2S- isopentenyl pyrophosphate.